The present invention relates to a method for transformation of anaerobic microorganisms and constructing shuttle vectors useful in the transformation. Specifically, the present invention relates to a method for transformation of bacteria belonging to the genus Clostridium Wl and to a family of shuttle vectors capable of transforming both anaerobic and aerobic species, such as species of Clostridium Wl and Escherichia coli (E. coli).
Anaerobic microorganisms are capable of growth in an environment lacking oxygen. Anaerobic microorganisms are usable in large-scale fermenters, where the ability to reproduce and to produce biological products in an environment lacking oxygen is perceived as an advantage. One reason that the ability to exhibit anaerobic growth is an advantage is that costly aeration apparatus for the fermentation vats need not exist when anaerobic organisms are grown in the vats. In addition, certain fermentation procedures exist where aeration is impossible. In these situations, it is necessary that an anaerobic microorganism be capable of carrying out the desired fermentation. Secondly, the possibility of contamination with aerobes is eliminated when an anaerobic environment is used. Thirdly, several anaerobic species are of special interest, both medically and commercially, for their abilities to produce toxins, use cellulose or pentoses as substrates, fix nitrogen, and produce useful bulk chemicals, such as acetone, butanol, acetic acid and ethanol as end products. For these reasons, methods have been sought which can be used to endow anaerobic microorganisms with additional genetic capabilities to produce novel biological products.
A method for endowing anaerobic microorganisms with the ability to produce useful biological products would be to transform the microorganism with plasmid DNA containing foreign genetic material, including genetic material from aerobic organisms, capable of directing the microorganism to produce the desired biological product. However, the inventors are unaware of any successful prior art transformation attempts that have resulted in a useful gene transfer system for anaerobic microorganisms. In fact, unsuccessful attempts at such a transformation using anaerobic microorganisms, such as that noted in Reid, S. J., E. R. Allcock, D. T. Jones, P. R. Woods, 1983, App. and Environmental Micro. 45: 305-307, have been published. In response to the commercial need for a method for transformation of anaerobic microorganisms, and in spite of previously reported unsuccessful attempts, the inventors have developed a method for transformation particularly applicable to anaerobic microorganisms more fully described below.